One third of the American adult population is currently considered obese with a greater risk of developing cardiovascular disease and diabetes. While much work has focused on studying the genetic and dietary factors of obesity, less is known at the cellular level. In particular, how lipids and proteins interact at the surface of lipid droplets to promote lipid storage remains unclear. Our long range objective is to identify the role of proteins and lipids in regulating lipid droplet metabolism. The current proposal focuses on the role of one protein, adipose differentiation-related protein (ADRP), in regulating lipid storage through interactions with specific lipid domains and/or proteins at the surface of lipid droplets. Our specific aims are: (1) Identify and characterize lipid domains within lipid droplets. The working hypothesis is that the lipid droplet monolayer contains not only phospholipids but also cholesterol and sphingolipids mat spontaneously organize into raft-like microdomains, similar to those found in the plasma membrane. We will use techniques established by our laboratory to isolate and characterize lipid domains in lipid droplets. (2) Characterize protein-lipid interactions at the surface of lipid droplets. The working hypothesis is that ADRP plays a key role in promoting lipid storage through interactions with lipids and proteins. While it is known that ADRP binds and colocalizes with fatty acids and cholesterol, to date no direct interaction between lipids or proteins has been shown. Multiphoton/confocal imaging and FRET will be used to study protein-lipid interactions. (3) Identify lipid binding motifs within ADRP. We will test the working hypothesis that ADRP contains lipid binding motifs that interact with specific lipids enriched in lipid domains to promote lipid storage by screening ADRP deletion mutants for ability to interact and bind with lipids. In summary, we propose to study the role of lipid domains in organizing a specific protein ADRP to function in lipid droplet metabolism. In the long term, determining the mechanism by which lipids are stored will impact how diseases associated with aberrant lipid metabolism including cardiovascular disease, diabetes, and obesity are treated and controlled.